K-088. Dual induction of zwf-1 Encoding Glucose-6-Phospahte Dehydrogenase by Intermediate of Entner-Douderoff Pathway and Oxidative Stress in Pseudomonas putida KT2440

J. Kim, W. Park;
Korea Univ., Seoul, REPUBLIC OF KOREA.

In Pseudomonas putida KT2440, there are three zwf homolog genes (zwf-1, zwf-2 and zwf-3) which encode glucose 6-phospahte dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phosphate to 6-phosphoglucolactone using either NAD or NADP. The transcription level of zwf-1gene dramatically increased when P. putida KT2440 was cultured in minimal medium containing either glucose or gluconate as a sole carbon source. However, the expression level of the zwf-1 gene was not detected in the presence of pyruvate or succinate. In case of zwf-2 or zwf-3, the expression was constitutively low in any condition. We constructed a GFP-based reporter fusion using the zwf-1 gene to study the activity of the zwf-1 gene promoter. The Pzwf-1activity highly increased in minimal medium supplemented either glucose or gluconate compared to pyruvate or succinate-minimal medium. A knock-out mutant of HexR (ΔHexR), a putative transcription regulator adjacent to zwf-1 gene, has constant Pzwf-1 activity in any condition. Electrophoretic mobility shift assay shows that the HexR protein binds the zwf-1 promoter region. Interestingly, the zwf-1 gene is also highly expressed in the presence of ROS producing materials such as paraquat, menadione, and arsenic. However, the induction level of zwf-2 and zwf-3 is nearly same their control condition. In the presence of menadione (1 mM) or paraquat (7μM), the ΔHexR strain shows the less sensitivity than WT. In E.coli, the zwf gene is activated by SoxRS regulon in oxidative stress condition. However, in Pseudomonas sp., the induction mechanism of the zwf-1 gene, expressed by oxidative stress, is unknown. Therefore, the zwf gene induction mechanisms by oxidative stress in P. putida KT2440 remains to be investigated. This work was supported by a NCRC grant (R15-2003-012-02002-0).